Bottom Line:
The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses.Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription.Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

Affiliation: State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACTAnalyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

Mentions:
To study the effect of SARS-CoV proteins on replication and transcription, individual viral proteins were co-expressed in trans with the SARS-CoV replicon. Renilla luciferase was employed as an internal control reporter to normalize the transfections among different wells (Fig. 4). Though most of the viral proteins did not exert significant impact on the replication and transcription of the replicon, the nsp3.1 containing X and SUD domains, and the RNA polymerase nsp12 (Fig. 4A), and nucleocapsid N (Fig. 4B) significantly increased the luciferase activity, whereas nsp3.2 containing the papain-like (PL) proteinase domain reduced the viral replication and transcription. Other proteins, such as nsp10/11, also showed a minor but significant increased expression. These results confirmed that the function of N gene fragment was not resulted from protein 9b that is internally nested in N sequence, as 9b showed no obvious impact on the replication and transcription of replicon when it was co-expressed (Fig. 4B). In addition, these results indicate that nucleocapsid protein, nsp3 and RNA polymerase may possess a dominant function in trans, while the PL proteinase is involved in important cis functions like cis-processing of the large viral polyprotein.

Mentions:
To study the effect of SARS-CoV proteins on replication and transcription, individual viral proteins were co-expressed in trans with the SARS-CoV replicon. Renilla luciferase was employed as an internal control reporter to normalize the transfections among different wells (Fig. 4). Though most of the viral proteins did not exert significant impact on the replication and transcription of the replicon, the nsp3.1 containing X and SUD domains, and the RNA polymerase nsp12 (Fig. 4A), and nucleocapsid N (Fig. 4B) significantly increased the luciferase activity, whereas nsp3.2 containing the papain-like (PL) proteinase domain reduced the viral replication and transcription. Other proteins, such as nsp10/11, also showed a minor but significant increased expression. These results confirmed that the function of N gene fragment was not resulted from protein 9b that is internally nested in N sequence, as 9b showed no obvious impact on the replication and transcription of replicon when it was co-expressed (Fig. 4B). In addition, these results indicate that nucleocapsid protein, nsp3 and RNA polymerase may possess a dominant function in trans, while the PL proteinase is involved in important cis functions like cis-processing of the large viral polyprotein.

Bottom Line:
The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses.Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription.Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

Affiliation:
State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACTAnalyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.